Background: Epithelial barrier integrity is critical for most gastrointestinal (GI) functions, including digestion of food, and absorption and secretion of nutrients. The gastrointestinal mucosal layer is very vulnerable to injury resulting in loss of barrier integrity, and this may all occur in critically ill veteran patients in response to surgical stress. Preventing it would likely have a major impact on outcomes and costs for all critically ill veteran populations, though the molecular controls that regulate barrier integrity are incompletely understood. This application will explore a promising approach to preserve, protect, and restore epithelial barrier function by manipulating the fundamental regulation of intestinal epithelial cells, while improving our overall understanding of their function and regulation. The bioactive sphingolipid sphingosine-1-phosphate (S1P) is a ubiquitous molecule produced intracellularly through a well characterized enzymatic cascade that can then regulate cell systems via cell surface receptors and known intracellular pathways. Manipulation of S1P has recently demonstrated significant effects on endothelial and epithelial cells in promoting important biological functions, such as survival of injured cells, and expression of barrier proteins. We have demonstrated in previously published work and our preliminary data that S1P has significant effects on intestinal epithelial cells and the intestinal barrier, both in physiologic and pathologic conditions. In this project we plan to build on our previous data supporting a potential therapeutic effect for S1P in accelerating the recovery of critically ill veterans in the intensive care unit. Objectives: We hypothesize that S1P protects the intestinal epithelial barrier in physiological and pathological surgical states as bot a 2nd messenger and extracellular ligand. To test this we will attempt to 1) Define the S1P signaling events related to intestinal epithelial barrier protein expression, 2) define the cellula mechanisms responsible for intestinal epithelial protection, 3) define the role of S1P in pathologic conditions both in vitro and in vivo. Methods: This proposal includes in vitro studies using cultured intestinal epithelial cells, ex vivo studies of tissues from mice and humans, and in vivo studies in mice. Findings: We have found intestinal epithelial cells, in response to S1P, show significant increases in barrier protein expression, proliferation and protection from apoptosis, and ultimately improved barrier function. S1P also has shown effects in stressful situations such as calcium free conditions or exposure to lipopolysaccharide. Status: Cells overexpressing Sphingosine Kinase 1, and expressing higher S1P content, show significantly increased proliferation, a process involving increased levels of c-Myc and a decrease in microRNA (mir) 542 among other findings. Mice exposed to S1P show improved barrier function in control states and during ischemia-reperfusion, and also decreased levels of mir 542. Impact: The proposed research addresses critical mechanisms in maintenance of an intact intestinal mucosal barrier, and in S1P, a potential therapy to preserve this function in critically ill patiens. Critical illness, particularly in surgical patients, is very common, and a high percentage of patients in the intensive care units incur stress-related injuries to their very vulnerable GI mucosa, leading to barrier dysfunction. Despite the medical advances of the past few decades, therapies for these gastro- intestinal injuries are, for the most part, conservative, as there is n current pharmacologic therapy available. This proposal explores such a pharmacologic therapy that could potentially prevent injury and loss of the intestinal epithelial cells and lead to improed outcomes of critically ill Veteran patients.